DNA Damage Response Processes Research Articles

DNA Double Strand Break and Response Fluorescent Assays: Choices and Interpretation.

Accurately characterizing DNA double-stranded breaks (DSBs) and understanding the DNA damage response (DDR) is crucial for assessing cellular genotoxicity, maintaining genomic integrity, and advancing gene editing technologies. Immunofluorescence-based techniques have proven to be invaluable for quantifying and visualizing DSB repair, providing valuable insights into cellular repair processes. However, the selection of appropriate markers for analysis can be challenging due to the intricate nature of DSB repair mechanisms, often leading to ambiguous interpretations. This comprehensively summarizes the significance of immunofluorescence-based techniques, with their capacity for spatiotemporal visualization, in elucidating complex DDR processes. By evaluating the strengths and limitations of different markers, we identify where they are most relevant chronologically from DSB detection to repair, better contextualizing what each assay represents at a molecular level. This is valuable for identifying biases associated with each assay and facilitates accurate data interpretation. This review aims to improve the precision of DSB quantification, deepen the understanding of DDR processes, assay biases, and pathway choices, and provide practical guidance on marker selection. Each assay offers a unique perspective of the underlying processes, underscoring the need to select markers that are best suited to specific research objectives.

Genetic insights into the age-specific biological mechanisms governing human ovarian aging

There is currently little evidence that the genetic basis of human phenotype varies significantly across the lifespan. However, time-to-event phenotypes are understudied and can be thought of as reflecting an underlying hazard, which is unlikely to be constant through life when values take a broad range. Here, we find that 74% of 245 genome-wide significant genetic associations with age at natural menopause (ANM) in the UK Biobank show a form of age-specific effect. Nineteen of these replicated discoveries are identified only by our modeling framework, which determines the time dependency of DNA-variant age-at-onset associations without a significant multiple-testing burden. Across the range of early to late menopause, we find evidence for significantly different underlying biological pathways, changes in the signs of genetic correlations of ANM to health indicators and outcomes, and differences in inferred causal relationships. We find that DNA damage response processes only act to shape ovarian reserve and depletion for women of early ANM. Genetically mediated delays in ANM were associated with increased relative risk of breast cancer and leiomyoma at all ages and with high cholesterol and heart failure for late-ANM women. These findings suggest that a better understanding of the age dependency of genetic risk factor relationships among health indicators and outcomes is achievable through appropriate statistical modeling of large-scale biobank data.

2-Hydroxy-3-methylanthraquinone inhibits homologous recombination repair in osteosarcoma through the MYC-CHK1-RAD51 axis

BackgroundOsteosarcoma is a malignant bone tumor that usually affects adolescents aged 15–19 y. The DNA damage response (DDR) is significantly enhanced in osteosarcoma, impairing the effect of systemic chemotherapy. Targeting the DDR process was considered a feasible strategy benefitting osteosarcoma patients. However, the clinical application of DDR inhibitors is not impressive because of their side effects. Chinese herbal medicines with high anti-tumor effects and low toxicity in the human body have gradually gained attention. 2-Hydroxy-3-methylanthraquinone (HMA), a Chinese medicine monomer found in the extract of Oldenlandia diffusa, exerts significant inhibitory effects on various tumors. However, its anti-osteosarcoma effects and defined molecular mechanisms have not been reported.MethodsAfter HMA treatment, the proliferation and metastasis capacity of osteosarcoma cells was detected by CCK-8, colony formation, transwell assays and Annexin V-fluorescein isothiocyanate/propidium iodide staining. RNA-sequence, plasmid infection, RNA interference, Western blotting and immunofluorescence assay were used to investigate the molecular mechanism and effects of HMA inhibiting osteosarcoma. Rescue assay and CHIP assay was used to further verified the relationship between MYC, CHK1 and RAD51.ResultsHMA regulate MYC to inhibit osteosarcoma proliferation and DNA damage repair through PI3K/AKT signaling pathway. The results of RNA-seq, IHC, Western boltting etc. showed relationship between MYC, CHK1 and RAD51. Rescue assay and CHIP assay further verified HMA can impair homologous recombination repair through the MYC-CHK1-RAD51 pathway.ConclusionHMA significantly inhibits osteosarcoma proliferation and homologous recombination repair through the MYC-CHK1-RAD51 pathway, which is mediated by the PI3K-AKT signaling pathway. This study investigated the exact mechanism of the anti-osteosarcoma effect of HMA and provided a potential feasible strategy for the clinical treatment of human osteosarcoma.Graphical

TEAD Proteins Associate With DNA Repair Proteins to Facilitate Cellular Recovery From DNA Damage

Transcriptional enhanced associate domain family members 1 to 4 (TEADs) are a family of four transcription factors and the major transcriptional effectors of the Hippo pathway. In order to activate transcription, TEADs rely on interactions with other proteins, such as the transcriptional effectors Yes-associated protein and transcriptional co-activator with PDZ-binding motif. Nuclear protein interactions involving TEADs influence the transcriptional regulation of genes involved in cell growth, tissue homeostasis, and tumorigenesis. Clearly, protein interactions for TEADs are functionally important, but the full repertoire of TEAD interaction partners remains unknown. Here, we employed an affinity purification mass spectrometry approach to identify nuclear interacting partners of TEADs. We performed affinity purification mass spectrometry experiment in parallel in two different cell types and compared a wildtype TEAD bait protein to a nuclear localization sequence mutant that does not localize to the nucleus. We quantified the results using SAINT analysis and found a significant enrichment of proteins linked to DNA damage including X-ray repair cross-complementing protein 5 (XRCC5), X-ray repair cross-complementing protein 6 (XRCC6), poly(ADP-ribose) polymerase 1 (PARP1), and Rap1-interacting factor 1 (RIF1). In cellular assays, we found that TEADs co-localize with DNA damage–induced nuclear foci marked by histone H2AX phosphorylated on S139 (γH2AX) and Rap1-interacting factor 1. We also found that depletion of TEAD proteins makes cells more susceptible to DNA damage by various agents and that depletion of TEADs promotes genomic instability. Additionally, depleting TEADs dampens the efficiency of DNA double-stranded break repair in reporter assays. Our results connect TEADs to DNA damage response processes, positioning DNA damage as an important avenue for further research of TEAD proteins.

The regulatory roles of the E3 ubiquitin ligase NEDD4 family in DNA damage response.

E3 ubiquitin ligases, an important part of ubiquitin proteasome system, catalyze the covalent binding of ubiquitin to target substrates, which plays a role in protein ubiquitination and regulates different biological process. DNA damage response (DDR) is induced in response to DNA damage to maintain genome integrity and stability, and this process has crucial significance to a series of cell activities such as differentiation, apoptosis, cell cycle. The NEDD4 family, belonging to HECT E3 ubiquitin ligases, is reported as regulators that participate in the DDR process by recognizing different substrates. In this review, we summarize recent researches on NEDD4 family members in the DDR and discuss the roles of NEDD4 family members in the cascade reactions induced by DNA damage. This review may contribute to the further study of pathophysiology for certain diseases and pharmacology for targeted drugs.

Transcriptional Profiling of the Candida albicans Response to the DNA Damage Agent Methyl Methanesulfonate

The infection of a mammalian host by the pathogenic fungus Candida albicans involves fungal resistance to reactive oxygen species (ROS)—induced DNA damage stress generated by the defending macrophages or neutrophils. Thus, the DNA damage response in C. albicans may contribute to its pathogenicity. Uncovering the transcriptional changes triggered by the DNA damage—inducing agent MMS in many model organisms has enhanced the understanding of their DNA damage response processes. However, the transcriptional regulation triggered by MMS remains unclear in C. albicans. Here, we explored the global transcription profile in response to MMS in C. albicans and identified 306 defined genes whose transcription was significantly affected by MMS. Only a few MMS-responsive genes, such as MGT1, DDR48, MAG1, and RAD7, showed potential roles in DNA repair. GO term analysis revealed that a large number of induced genes were involved in antioxidation responses, and some downregulated genes were involved in nucleosome packing and IMP biosynthesis. Nevertheless, phenotypic assays revealed that MMS-induced antioxidation gene CAP1 and glutathione metabolism genes GST2 and GST3 showed no direct roles in MMS resistance. Furthermore, the altered transcription of several MMS—responsive genes exhibited RAD53—related regulation. Intriguingly, the transcription profile in response to MMS in C. albicans shared a limited similarity with the pattern in S. cerevisiae, including COX17, PRI2, and MGT1. Overall, C. albicans cells exhibit global transcriptional changes to the DNA damage agent MMS; these findings improve our understanding of this pathogen’s DNA damage response pathways.

Aldehyde Dehydrogenase 1B1 Is Implicated in DNA Damage Response in Human Colorectal Adenocarcinoma

Aldehyde dehydrogenase 1B1 (ALDH1B1) has been correlated with colorectal tumorigenesis and is considered a potential biomarker for colon cancer. Its expression has been associated with attenuation of the cell cycle in the G2/M phase and resistance to DNA damaging agents. The present study examines the role of ALDH1B1 in DNA damage response (DDR) in human colorectal adenocarcinoma. To this end, we utilized an isogenic HT29 cell line pair differing in the expression of ALDH1B1. The overexpression of ALDH1B1 was related to the translational upregulation of the total and phosphorylated (at ser15) p53. Comet and apoptosis assays revealed that the expression of ALDH1B1 protected HT29 cells from etoposide-induced DNA damage as well as apoptosis, and its overexpression led to increased constitutive phosphorylation of H2AX (at ser139). Furthermore, the expression profile of a variety of DNA damage signaling (DDS)-related genes was investigated by utilizing the RT2 profiler™ PCR array. Our results demonstrated that ALDH1B1 triggered a transcriptional activation of several DNA repair-related genes (MRE11A, PMS1, RAD18 and UNG). Finally, Spearman’s rank correlation coefficient analysis in 531 publicly available colorectal adenocarcinoma clinical samples indicated the statistically significant positive correlation between ALDH1B1 and DDR and repair genes or proteins, such as APEX1, FEN1, MPG, UNG, XRCC1, DDB1, XPC, CIB1, MRE11, PRKDC, RAD50, RAD21, TP53BP1, XRCC6 and H2AX. Collectively, our results suggest that ALDH1B1 may play an essential role in the DDR and DNA repair processes. Further studies on ALDH1B1 will elucidate its precise role in DDR.

NEDD9 Restrains dsDNA Damage Response during Non-Small Cell Lung Cancer (NSCLC) Progression.

Simple SummaryAltered DNA damage response (DDR) contributes to numerous processes during the progression of tumors, such as genomic instability, the emergence of neoantigens, aberrations in cell-cell signaling, and acquired tumor resistance to DNA damaging agents, such as platinating agents and irradiation. This study describes a novel role for the scaffolding protein NEDD9 in regulating DDR signaling and characterizes its effects on sensitivity to DNA damaging therapies in a non-small cell lung cancer (NSCLC) setting. Our data demonstrate that NEDD9 depletion is capable of upregulating ATM-CHK2 signaling, shifting NEDD9 depleted cells towards a more mesenchymal phenotype and elevated sensitivity to UV irradiation. Immunohistochemical analysis of the cohort of human NSCLC samples revealed an association between reduced NEDD9 protein expression and a decrease in overall (OS) survival of NSCLC patients.DNA damaging modalities are the backbone of treatments for non-small cell lung cancer (NSCLC). Alterations in DNA damage response (DDR) in tumor cells commonly contribute to emerging resistance to platinating agents, other targeted therapies, and radiation. The goal of this study is to identify the previously unreported role of NEDD9 scaffolding protein in controlling DDR processes and sensitivity to DNA damaging therapies. Using a siRNA-mediated approach to deplete NEDD9 in a group of human and murine KRAS/TP53-mutant NSCLC cell lines, coupled with a set of cell viability and clonogenic assays, flow cytometry analysis, and Western blotting, we evaluated the effects of NEDD9 silencing on cellular proliferation, DDR and epithelial-to-mesenchymal transition (EMT) signaling, cell cycle, and sensitivity to cisplatin and UV irradiation. Using publicly available NSCLC datasets (TCGA) and an independent cohort of primary NSCLC tumors, subsequent in silico and immunohistochemical (IHC) analyses were performed to assess relevant changes in NEDD9 RNA and protein expression across different stages of NSCLC. The results of our study demonstrate that NEDD9 depletion is associated with the increased tumorigenic capacity of NSCLC cells. These phenotypes were accompanied by significantly upregulated ATM-CHK2 signaling, shifting towards a more mesenchymal phenotype in NEDD9 depleted cells and elevated sensitivity to UV-irradiation. IHC analyses revealed an association between reduced NEDD9 protein expression and a decrease in overall (OS) and progression-free survival (PFS) of the NSCLC patients. These data, for the first time, identified NEDD9 as a negative regulator of ATM kinase activity and related DDR signaling in numerous KRAS/TP53 mutated NSCLC, with its effects on the regulation of DDR-dependent EMT signaling, sensitivity to DNA damaging modalities in tumor cells, and the survival of the patients.

Targeting the Homologous Recombination Pathway in Cancer With a Novel Class of RAD51 Inhibitors.

Targeting DNA damage response (DDR) pathway has been proposed as an approach for amplifying tumor-specific replicative lesions. RAD51 plays a central role in the DDR process, and thus represents a promising anti-tumor target. We here report the discovery of a series of next generation RAD51 inhibitors that can prevent RAD51 foci formation. The lead compounds dramatically impaired human cancer cell growth, induced cell cycle arrest in S-phase, and resulted in elevated γH2AX. Furthermore, cancer cells became sensitized to chemotherapy and other DDR inhibitors. Dosed either as a single agent or in combination with cisplatin, the compounds significantly inhibited tumor growth in vivo. By upregulating ATR-CHK1 signaling, the RAD51 inhibitors increased surface PD-L1 levels in various tumor cells, suggesting a potential combination of RAD51 inhibitors with PD-1/PD-L1 blockade. Overall, our findings provide the preclinical rationale to explore RAD51 inhibitors as monotherapy or in combination with chemotherapy, immunotherapy or DDR-targeting therapy in cancer treatment.

Ionizing radiation-induced DNA damage responses affect cell compressibility.

Radiotherapy is an important treatment modality for cancer patients. Ionizing radiation kills cancer cells by inducing DNA damage. However, it remains unclear how this process affects the biomechanical properties of cells and how cellular mechanics affect DNA damage and repair. In this study, the effects of ionizing radiation on cell mechanics were investigated by precisely measuring the compressibility of cells in suspension using an in-house developed microfluidic device. We found that cell compressibility significantly increased after irradiation, depending on cell type and radiation dose. Radiation-induced DNA damage response (DDR) is an important process that alters cell mechanics, and this association was confirmed experimentally by inhibiting the DDR process. Furthermore, the mechanisms involved in changes in cell compressibility were investigated from the perspective of the nucleus and cytoplasm. Experiments showed that radiation reduced H3K9me3 staining intensity but had no effect on F-actin. This suggested that chromatin decondensation caused by radiation-induced DDR is the primary cause of changes in cell compressibility after radiation. The effects of cell mechanics on radiation-induced DNA damage were also investigated. The addition of blebbistatin reduced cytoskeletal forces on the nucleus, which resulted in a reduction in radiation-induced DNA damage. Collectively, this study elucidated reciprocal mechanisms of radiation-induced DNA damage and cell mechanics.

Beyond BRCA: The Emerging Significance of DNA Damage Response and Personalized Treatment in Pancreatic and Prostate Cancer Patients.

The BRCA1/2 germline and/or somatic pathogenic variants (PVs) are key players in the hereditary predisposition and therapeutic response for breast, ovarian and, more recently, pancreatic and prostate cancers. Aberrations in other genes involved in homologous recombination and DNA damage response (DDR) pathways are being investigated as promising targets in ongoing clinical trials. However, DDR genes are not routinely tested worldwide. Due to heterogeneity in cohort selection and dissimilar sequencing approaches across studies, neither the burden of PVs in DDR genes nor the prevalence of PVs in genes in common among pancreatic and prostate cancer can be easily quantified. We aim to contextualize these genes, altered in both pancreatic and prostate cancers, in the DDR process, to summarize their hereditary and somatic burden in different studies and harness their deficiency for cancer treatments in the context of currently ongoing clinical trials. We conclude that the inclusion of DDR genes, other than BRCA1/2, shared by both cancers considerably increases the detection rate of potentially actionable variants, which are triplicated in pancreatic and almost doubled in prostate cancer. Thus, DDR alterations are suitable targets for drug development and to improve the outcome in both pancreatic and prostate cancer patients. Importantly, this will increase the detection of germline pathogenic variants, thereby patient referral to genetic counseling.

Molecular mechanisms in governing genomic stability and tumor suppression by the SETD2 H3K36 methyltransferase.

Epigenetic dysregulation is an important contributor to carcinogenesis. This is not surprising, as chromatin-genomic DNA organized around structural histone scaffolding-serves as the template on which occurs essential nuclear processes, such as transcription, DNA replication and DNA repair. Histone H3 lysine 36 (H3K36) methyltransferases, such as the SET-domain 2 protein (SETD2), have emerged as critical tumor suppressors. Previous work on mammalian SETD2 and its counterpart in model organisms, Set2, has highlighted the role of this protein in governing genomic stability through transcriptional elongation and splicing, as well as in DNA damage response processes and cell cycle progression. A compendium of SETD2 mutations have been documented, garnered from sequenced cancer patient genome data, and these findings underscore the cancer-driving properties of SETD2 loss-of-function. In this review, we consolidate the molecular mechanisms regulated by SETD2/Set2 and discuss evidence of its dysregulation in tumorigenesis. Insight into the genetic interactions that exist between SETD2 and various canonical intracellular signaling pathways has not only empowered pharmacological intervention by taking advantage of synthetic lethality but underscores SETD2 as a druggable target for precision cancer therapy.

Molecular mechanisms involved in DNA repair in human cancers: An overview of PI3k/Akt signaling and PIKKs crosstalk

The cellular genome is frequently subjected to abundant endogenous and exogenous factors that induce DNA damage. Most of the Phosphatidylinositol 3-kinase-related kinases (PIKKs) family members are activated in response to DNA damage and are the most important DNA damage response (DDR) proteins. The DDR system protects the cells against the wrecking effects of these genotoxicants and repairs the DNA damage caused by them. If theDNA damage issevere, such as when DNA is the goal of chemo-radiotherapy, the DDR drives cells toward cell cycle arrest and apoptosis. Some intracellular pathways, such as PI3K/Akt, which is overactivated in most cancers, could stimulate the DDR process and failure of chemo-radiotherapy with the increasing repair of damaged DNA. This signaling pathway induces DNA repair through the regulation of proteins that are involved in DDR like BRCA1, HMGB1, and P53. In this review, we will focus on the crosstalk of the PI3K/Akt and PIKKs involved in DDR and then discuss current achievements in the sensitization of cancer cells to chemo-radiotherapy by PI3K/Akt inhibitors.

Genetic insights into biological mechanisms governing human ovarian ageing.

Reproductive longevity is essential for fertility and influences healthy ageing in women1,2, but insights into its underlying biological mechanisms and treatments to preserve it are limited. Here we identify 290 genetic determinants of ovarian ageing, assessed using normal variation in age at natural menopause (ANM) in about 200,000 women of European ancestry. These common alleles were associated with clinical extremes of ANM; women in the top 1% of genetic susceptibility have an equivalent risk of premature ovarian insufficiency to those carrying monogenic FMR1 premutations3. The identified loci implicate a broad range of DNA damage response (DDR) processes and include loss-of-function variants in key DDR-associated genes. Integration with experimental models demonstrates that these DDR processes act across the life-course to shape the ovarian reserve and its rate of depletion. Furthermore, we demonstrate that experimental manipulation of DDR pathways highlighted by human genetics increases fertility and extends reproductive life in mice. Causal inference analyses using the identified genetic variants indicate that extending reproductive life in women improves bone health and reduces risk of type 2 diabetes, but increases the risk of hormone-sensitive cancers. These findings provide insight into the mechanisms that govern ovarian ageing, when they act, and how they might be targeted by therapeutic approaches to extend fertility and prevent disease.

Detection of Genes in Arabidopsis thaliana L. Responding to DNA Damage from Radiation and Other Stressors in Spaceflight

Ionizing radiation present in extraterrestrial environment is an important factor that affects plants grown in spaceflight. Pearson correlation-based gene regulatory network inferencing from transcriptional responses of the plant Arabidopsis thaliana L. grown in real and simulated spaceflight conditions acquired by GeneLab, followed by topological and spectral analysis of the networks is performed. Gene regulatory subnetworks are extracted for DNA damage response processes. Analysis of radiation-induced ATR/ATM protein–protein interactions in Arabidopsis reveals interaction profile similarities under low radiation doses suggesting novel mechanisms of DNA damage response involving non-radiation-induced genes regulating other stress responses in spaceflight. The Jaccard similarity index shows that the genes AT2G31320, AT4G21070, AT2G46610, and AT3G27060 perform similar functions under low doses of radiation. The incremental association Markov blanket method reveals non-radiation-induced genes linking DNA damage response to root growth and plant development. Eighteen radiation-induced genes and sixteen non-radiation-induced gene players have been identified from the ATR/ATM protein interaction complexes involved in heat, salt, water, osmotic stress responses, and plant organogenesis. Network analysis and logistic regression ranking detected AT3G27060, AT1G07500, AT5G66140, and AT3G21280 as key gene players involved in DNA repair processes. High atomic weight, high energy, and gamma photon radiation result in higher intensity of DNA damage response in the plant resulting in elevated values for several network measures such as spectral gap and girth. Nineteen flavonoid and carotenoid pigment activations involved in pigment biosynthesis processes are identified in low radiation dose total light spaceflight environment but are not found to have significant regulations under very high radiation dose environment.

DNA Damage Response in the Adaptive Arm of the Immune System: Implications for Autoimmunity.

In complex environments, cells have developed molecular responses to confront threats against the genome and achieve the maintenance of genomic stability assuring the transfer of undamaged DNA to their progeny. DNA damage response (DDR) mechanisms may be activated upon genotoxic or environmental agents, such as cytotoxic drugs or ultraviolet (UV) light, and during physiological processes requiring DNA transactions, to restore DNA alterations that may cause cellular malfunction and affect viability. In addition to the DDR, multicellular organisms have evolved specialized immune cells to respond and defend against infections. Both adaptive and innate immune cells are subjected to DDR processes, either as a prerequisite to the immune response, or as a result of random endogenous and exogenous insults. Aberrant DDR activities have been extensively studied in the immune cells of the innate arm, but not in adaptive immune cells. Here, we discuss how the aberrant DDR may lead to autoimmunity, with emphasis on the adaptive immune cells and the potential of therapeutic targeting.

O2: TOWARDS A LIVING BIOBANK OF SURGICALLY-RELEVANT 3-DIMENSIONAL GLIOBLASTOMA STEM CELL MODELS TO EVALUATE NOVEL THERAPEUTICS AND INTERROGATE INTRATUMOURAL HETEROGENEITY

Abstract Introduction Glioblastoma is the most common cancer arising within the brain. Despite surgery, followed by DNA-damaging chemoradiotherapy, average survival remains between 12-15 months. Unacceptable survival rates underline the need to develop preclinical research models which recapitulate features underpinning therapeutic resistance in patients, such as intratumoural heterogeneity and treatment resistant glioblastoma stem cell (GSC) subpopulations which demonstrate elevated DNA damage response (DDR) activity. Method Tumour specimens from patients were used to generate 2D and 3D scaffold-based GSC models, with a range of preclinical survival and molecular assays used to interrogate cancer biology and assess therapeutic responses. Result We have developed a ‘living biobank’ of 20+ ex-vivo GSC models which reflect key clinicopathological diversity. These models include residual disease models based on careful macrodissection of rare en-blocpartial lobectomy specimens to liberate parallel GSC lines from the tumour core and adjacent infiltrated brain, to represent cells typically left behind after surgery. Therapeutic strategies targeting fundamental DDR processes demonstrate preclinical efficacy, for example dual inhibition of ATR and the FA DNA damage repair pathways elicits profound radiosensitisation (sensitiser enhancement ratio of 3.23 (3.03-3.49, 95%-CI)) with evidence of delayed DNA damage repair on single-cell gel electrophoresis. Finally, characterisation of our surgically-relevant resected and residual models reveals numerous divergent properties including elevated stem cell marker expression in residual models (p=0.0021), which may partially explain treatment resistance in disease left behind after surgery. Conclusion Our living biobank represents a useful resource for preclinical glioblastoma research and demonstrates the value of partnership between surgeons and laboratory-based scientists. Take-home message Our living biobank represents a useful resource for preclinical glioblastoma research and demonstrates the value of partnership between surgeons and laboratory-based scientists.

Alteration of DNA Damage Response Causes Cleft Palate.

Cleft palate is one of the most common craniofacial birth defects, however, little is known about how changes in the DNA damage response (DDR) cause cleft palate. To determine the role of DDR during palatogenesis, the DDR process was altered using a pharmacological intervention approach. A compromised DDR caused by a poly (ADP-ribose) polymerase (PARP) enzyme inhibitor resulted in cleft palate in wild-type mouse embryos, with increased DNA damage and apoptosis. In addition, a mouse genetic approach was employed to disrupt breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2), known as key players in DDR. An ectomesenchymal-specific deletion of Brca1 or Brca2 resulted in cleft palate due to attenuation of cell survival. This was supported by the phenotypes of the ectomesenchymal-specific Brca1/Brca2 double-knockout mice. The cleft palate phenotype was rescued by superimposing p53 null alleles, demonstrating that the BRCA1/2–p53 DDR pathway is critical for palatogenesis. Our study highlights the importance of DDR in palatogenesis.

Exploring microRNA Signatures of DNA Damage Response Using an Innovative System of Genotoxic Stress in Medicago truncatula Seedlings.

One of the challenges that living organisms face is to promptly respond to genotoxic stress to avoid DNA damage. To this purpose, all organisms, including plants, developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms and need to be finely regulated. In this scenario, microRNAs (miRNAs) are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells whereas studies in plants are still scarce. To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC), targeting distinct components of DDR, namely topoisomerase I (TopI) and tyrosyl-DNA phosphodiesterase 1 (TDP1), were used. Phenotypic (germination percentage and speed, seedling growth) and molecular (cell death, DNA damage, and gene expression profiles) analyses demonstrated that the imposed treatments impact DDR. Our results show that these treatments do not influence the germination process but rather inhibit seedling development, causing an increase in cell death and accumulation of DNA damage. Moreover, treatment-specific changes in the expression of suppressor of gamma response 1 (SOG1), master-regulator of plant DDR, were observed. Additionally, the expression of multiple genes playing important roles in different DNA repair pathways and cell cycle regulation were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identified from our previous bioinformatics approaches as putatively targeting genes involved in DDR processes were investigated alongside their targets. The obtained results indicate that under most conditions when a miRNA is upregulated the corresponding candidate target gene is downregulated, providing an indirect evidence of miRNAs action over these targets. Hence, the present study extends the present knowledge on the information available regarding the roles played by miRNAs in the post-transcriptional regulation of DDR in plants.

Crosstalk between Different DNA Repair Pathways Contributes to Neurodegenerative Diseases.

Simple SummaryConstant exposure to endogenous and environmental factors induces oxidative stress and DNA damage. Rare brain disorders caused by defects in DNA repair and DNA damage response (DDR) signaling establish that failure to process DNA damage may lead to neurodegeneration. In this review, we present mechanisms that link DDR with neurodegeneration in these disorders and discuss their relevance for common age-related neurodegenerative diseases (NDDs). Moreover, we highlight recent insight into the crosstalk between the DDR and other cellular processes known to be disturbed during NDDs.Genomic integrity is maintained by DNA repair and the DNA damage response (DDR). Defects in certain DNA repair genes give rise to many rare progressive neurodegenerative diseases (NDDs), such as ocular motor ataxia, Huntington disease (HD), and spinocerebellar ataxias (SCA). Dysregulation or dysfunction of DDR is also proposed to contribute to more common NDDs, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Amyotrophic Lateral Sclerosis (ALS). Here, we present mechanisms that link DDR with neurodegeneration in rare NDDs caused by defects in the DDR and discuss the relevance for more common age-related neurodegenerative diseases. Moreover, we highlight recent insight into the crosstalk between the DDR and other cellular processes known to be disturbed during NDDs. We compare the strengths and limitations of established model systems to model human NDDs, ranging from C. elegans and mouse models towards advanced stem cell-based 3D models.